Effect of GABAergic inhibition of dendritic spines on synaptic pruning in the medial prefrontal cortex during adolescence

青春期，内侧前额叶皮质（mPFC）中的树枝状棘经历突触修剪， 这是精神分裂症中过度的一个过程，被认为是认知障碍的基础。尽管 重要的是，mPFC中突触修剪的机制尚不清楚。我们的初步 研究结果表明，突触外α4βδ GABAA受体（GABARs），短暂出现在 在青春期开始（~PND 35）的脊髓中，持续10 d，触发mPFC中的青春期突触修剪。这些 受体产生削弱NMDA受体活化的分流抑制。我们之前的发现 在CA 1海马中，这些受体产生的抑制作用降低了 Rho-GEF脊柱蛋白Kalirin-7，脊柱维持所必需的，通过这种减少的NMDA受体 活动我们将检验这一假设，即对脊柱蛋白的类似影响在青少年中发挥作用。 mPFC中的修剪。拟议的目标将使用多种技术，包括体内双光子 成像、电生理学、药理学和免疫细胞化学测定。为此我们将 在青春期（~PND 35-44）长期操纵α4βδ功能，并在8岁时量化脊柱密度 和12周龄。初步数据显示，脊柱密度在第3层和第5层都有所下降， 在+/+而不是α4-/-小鼠中，青春期，涉及α4βδ GABAR。因为突触GABA能输入 靶向皮质中的脊柱，还将检查α1β2γ2 GABAR的作用。这些实验 将使用急性药理学（激动剂、调节剂）和遗传操作（整体/条件性/局部 敲低）α4βδ和α1β2γ2，以探索这些受体在调节棘密度和 使用高尔基体和电生理学技术评估形态学。为了探索这种影响， 修剪电路和行为可塑性，在体内双光子技术结合多， 神经元记录将用于对Ca++信号进行成像并记录由神经元产生的活动。 整个青春期和GABAR后清醒小鼠对视觉刺激的反应 调制/击倒。因此，我们将评估青春期α4βδ GABAR和GABAR的作用。 生成修剪对电路的功能，包括功率和同步的theta和gamma 振荡以及电路活动/合奏同步和mPFC依赖性学习 任务拟议研究的结果将直接解决GABA能的功能作用， 抑制青春期树突棘，也将提供突触过程的机制， 修剪，以及探索在这个过程中的改变的功能结果。这些结果是相关的 对于精神分裂症，其中过度突触修剪和工作记忆受损的报道， 关联降低了视觉诱发伽马振荡的功率/同步， 报道了δ基因的核苷酸多态性，表明存在遗传联系。

Dendritic spines undergo synaptic pruning in the medial prefrontal cortex (mPFC) during adolescence, a process which is excessive in schizophrenia, believed to underlie cognitive impairment. Despite its importance, the mechanism underlying synaptic pruning in the mPFC is not known. Our preliminary findings suggest that extrasynaptic α4βδ GABAA receptors (GABARs), which transiently emerge on the spine at puberty onset (~PND 35) for 10d, trigger adolescent synaptic pruning in the mPFC. These receptors generate a shunting inhibition which impairs NMDA receptor activation. Our previous findings in CA1 hippocampus established that the inhibition generated by these receptors reduces expression of the Rho-GEF spine protein Kalirin-7, necessary for spine maintenance, via this reduced NMDA receptor activity. We will test the hypothesis that similar effects on spine proteins play a role in adolescent pruning in mPFC. The proposed aims will use multiple techniques, including in vivo two-photon imaging, electrophysiological, pharmacological, and immunocytochemical assays. To this end, we will chronically manipulate α4βδ function during adolescence (~PND 35-44) and quantify spine density at 8 and 12 wks of age. Preliminary data shows that spine density is decreased in both layer 3 and 5 post- pubertally in +/+ but not α4-/- mice, implicating α4βδ GABARs. Because synaptic GABAergic input targets the spine in the cortex, the role of α1β2γ2 GABARs will also be examined. These experiments will use acute pharmacological (agonists, modulators) and genetic manipulation (global/conditional/local knock-down) of α4βδ and α1β2γ2 to explore the role of these receptors in regulating spine density and morphology assessed using Golgi and electrophysiological techniques. To explore the effect of this pruning on circuit and behavioral plasticity, in vivo two-photon techniques in combination with multi- neuron recording will be used to image the Ca++ signals and record activity generated by neuronal ensembles in response to visual stimuli in the awake mouse across adolescence and after GABAR modulation/knock-down. We will thus assess the effect of pubertal α4βδ GABARs and GABAR- generated pruning on circuit function, including the power and synchronization of theta and gamma oscillations as well as on circuit activity/synchronization of ensembles and mPFC-dependent learning tasks. The findings from the proposed studies will directly address the functional role of GABAergic inhibition on dendritic spines at puberty, and will also provide mechanisms for the process of synaptic pruning, as well as explore functional outcomes of alterations in this process. These results are relevant for schizophrenia where excessive synaptic pruning and impaired working memory are reported in association reduced power/sychronization of visually-evoked gamma oscillations, and where single nucleotide polymorphisms of the δ gene are reported, suggesting a genetic link.